Diindolylmethane formulations for the treatment of leiomyomas

ABSTRACT

The present invention relates to compositions and methods for treating or preventing leiomyomas by administration of diindolylmethane and diindolylmethane-related indoles. The present invention also relates to compositions and methods for treating or preventing leiomyomas by administration of diindolylmethane in combination with an EGFR antagonist. The methods provide non-invasive treatments for leiomyomas.

This application claims priority to U.S. Provisional Application No.60/569,478, filed on May 6, 2004, which is incorporated herein byreference in its entirety.

1. FIELD OF THE INVENTION

The present invention relates to compositions and methods for treatingor preventing leiomyomas by administration of diindolylmethane or adiindolylmethane-related indole. The present invention also relates tocompositions and methods for treating or preventing leiomyomas byadministration of diindolylmethane or a diindolylmethane-related indolein combination with an EGFR antagonist. The methods provide non-invasivetreatments for leiomyomas.

2. BACKGROUND OF THE INVENTION

2.1. Clinical Importance of Uterine Leiomyomas

Leiomyomas are benign soft-tissue tumors that arise from smooth muscletissue. Leiomyomas of the uterus are the most common, abnormal pelvicgrowth diagnosed in women. Leiomyomas are also called “myomas” and“uterine fibroids”. As benign, non-cancerous, growths arising fromuterine smooth muscle (myometrium), leiomyomas are unrelated to thecommon forms of uterine cancer and uterine cervical cancer whichtypically arise from the uterine lining (endometrium) or cervicalepithelium, respectively. In the United States, leiomyomas occur in themajority of all women (Flake et al., 2003, Environ Health Perspect.111:1037-54). Leiomyomas are particularly prevalent in African-Americanwomen, who report a 3 times greater rate of occurrence than Caucasianwomen (Marshall et al., 1997, Obstet. Gynecol. 90:967-73). Althoughrare, leiomyomas also develop outside the uterus in both women and men.Extra-uterine sites of leiomyoma occurrence include the nipple,esophagus, scrotum, and seminal vesicles which all possess smooth muscle(Farman, 1975, S Afr Med J. 49:1333-40). In addition, rare, familialsyndromes, including diffuse leiomyomatosis and Alport Syndrome, areassociated with recurrent leiomyomas in multiple anatomic sites. Smoothmuscle tumors are also common in the avian species, typically involvingthe oviduct, an analogous structure to the mammalian uterus. Japanesequail (Coturnix cturnix japonica) and the common chicken hen (Gallusdomesticus) are especially affected, with prevalences varying up to 60%(Foster et al., 1989, Poult Sci. 68:1447-53; and Anjum et al., 1988, ResVet Sci. 45:341-8). Despite their frequency, the pathobiology ofleiomyomas remains poorly understood.

In women, leiomyomas contribute to a spectrum of symptoms includingheavy, irregular, and prolonged menstrual bleeding and anemia.Leiomyomas may also cause pelvic discomfort, and bowel and bladderdysfunction from pressure on these adjacent structures. Leiomyomas havealso been associated with infertility and recurrent abortion. Thepresence of leiomyomas can interfere with normal labor during the birthprocess, necessitating cesarean section. In addition to abnormal labor,leiomyomas restrict normal intra-uterine growth and predispose topostpartum hemorrhage secondary to uterine atony.

Histologically, leiomyomas arise from smooth muscle, show benign cellstructure, and are distinct and unrelated to precancerous and cancerouscell growth affecting the uterine lining or endometrium. Leiomyomasarise from genetically similar clones of uterine smooth muscle cells,and they grow under the influence of local growth factors and in thepresence of sex hormones. Evidence supports a more importantcontribution of progesterone than estrogen to myoma growth (Rein, 2000,Environ Health Perspect. 108 Suppl 5:791-3). The contribution ofprogesterone and estrogen to leiomyoma growth is uncertain, however,based on the observation that leiomyomas often recede in size duringpregnancy, which is a time of high circulating estrogen and progesteronelevels (Strobelt et al., 1994, J Ultrasound Med. 13:399-401). Adding tothe uncertainty of the role of estrogen in myoma etiology, the clinicaluse of tamoxifen, an estrogen antagonist drug, has been associated withincreased, not decreased, growth of leiomyomas (Schwartz et al., 1998, JUltrasound Med. 17:699-703). Typically, leiomyomas in women appear aftermenarche, proliferate and grow during the reproductive years, andstabilize or regress after menopause. The diagnosis of leiomyomas isbased on patient signs and symptoms, followed by physical and pelvicexamination, demonstrating a pelvic mass, and confirmation bytrans-abdominal or transvaginal ultrasonic visualization. The etiologyand hormonal contributions to leiomyomas remain poorly understood.

Study of the biochemistry of uterine leiomyomas has shown that myomatissue preferentially metabolizes estrogen to 4-hydroxy estrogenmetabolites, demonstrating a different pattern of estrogen metabolismthan surrounding normal myometrial smooth muscle (Liehr et al., 1995,Proc Natl Acad Sci USA 92:9220-4). Biochemical comparison of theextra-cellular matrix of leiomyoma tissue compared to surrounding normaltissue reveals differences in the composition and structure of collagenwhich may contribute to abnormal growth (Berto et al., 2003, BiochimBiophys Acta. 1619:98-112.)

Other than surgery, there are few safe and effective treatment optionsavailable to women with symptomatic leiomyomas. Gonadotropin-releasinghormone agonists (GnRH-a) inhibit steroidogenesis and inducepharmacologic menopause. Through this mechanism, GnRH-a's can reduceleiomyoma volume by 50% in 3 to 6 months. However, because these agentscause severe menopausal symptoms and the risk of estrogen-deficiencyrelated bone loss (osteoporosis), these drugs cannot be used forprolonged periods of time. Moreover, leiomyomas tend to regrow aftercessation of GnRH-a therapy. As a result, GnRH-a treatment, independentof subsequent surgery, is not recognized as an effective, long-termtreatment of leiomyomas.

The surgical procedures for treatment of uterine leiomyomas aremyomectomy and hysterectomy (Nair, 2003, Ann Acad Med. Singapore.32:615-23). Myomectomy, done either through a laparotomy or laparoscopy,is performed to remove the leiomyoma and conserve the uterus. This isusually attempted in young women who may desire future pregnancy.Unfortunately, myomectomy is followed by extensive pelvic adhesions thatthemselves can reduce future fertility. Additionally, if the leiomyomapenetrates the uterine cavity, any future pregnancy after myomectomycarries an increased risk of uterine rupture and delivery has to beaccomplished by cesarean section.

Hysterectomy remains the definitive surgical treatment for leiomyomas.Symptomatic uterine leiomyomas account for approximately one third ofall hysterectomies performed among middle-aged women (Nair, 2003, AnnAcad Med. Singapore. 32:615-23). The impact of this surgical approach toleiomyomas is extremely costly considering the long postoperative timeand recuperation away from work. There are also well known complicationsto hysterectomy. These complications include postoperative hemorrhage,fever, or injury to adjacent organs.

Two recent modalities have been developed for less extremeinterventional treatment of uterine fibroids: myolysis and uterineartery embolization (Lefebvre et al., 2003, J Obstet Gynaecol Can.25:396-418). Myolysis refers to the technique of disrupting orabolishing the blood supply to the fibroid causing shrinkage by usingbipolar or monopolar electrosurgery. It is only applicable if there areless than three fibroids present and/or the largest one measures lessthan 10 cm in diameter. The procedure is also not recommended for womenwho wish to get pregnant, since the risk of uterine rupture is high.Uterine artery embolization (UAE) is a procedure done by radiologistswith the objective of eliminating the blood supply to leiomyoma tissue.After some months following the treatment, UAE typically results in anaverage reduction of myoma tissue volume of about 50%. However, acutelycausing the death of myoma tissue due to arterial blockage has sideeffects. The procedure can be followed by severe pain requiringhospitalization. Some concern about future fertility has been raisedfollowing UAE, as well as lack of ability to treat all leiomyomaspresent. A portion of women undergoing uterine artery embolism developsubsequent amenorrhea and menopause due to inadvertent impairment ofovarian function (Spies et al., 2002, Obstet. Gynecol. 100:873-80).

For women with symptomatic leiomyomas, who wish to preserve fertility oravoid surgery, more conservative, more effective, and safer methods ofmedical treatment are needed. Ideally, improved medical treatments tomanage leiomyomas will preserve an intact uterus, better preservefertility, and, as adjunct treatments, improve the safety and long-termefficacy of more conservative treatment modalities such as UAE andlaparoscopic myomectomy.

2.2. Biologic Activities of Cruciferous Indoles

Cruciferous vegetables contain a family of plant protective compoundscalled glucobrassicins which give rise to active compounds with theindole molecular ring, exemplified by indole-3-carbinol (I3C). However,I3C is highly unstable in water and acid. When given orally, I3Cgenerates a number of gastric reaction products with a variety ofbiologic actions (De Kruif et al., 1991, Chem Biol Interact; 80:303-15).These products are highly enzyme inducing and associated with both theinactivation and activation of carcinogens. As such, the use of I3C hasbeen associated with both the growth inhibition and growth promotion inexperimental cancers. In addition, unwanted enzyme induction by I3Creaction products following oral I3C use may alter the metabolism ofother drugs, steroid hormones including estrogen, and contraceptivesraising safety concerns. Oral use of I3C in humans has been shown toincrease production of the 4-hydroxy estrogen metabolites previouslyassociated with leiomyoma tissue (Michnovicz et al., 1997, J Natl CancerInst. 89:718-23). In addition, I3C's use is associated with a number ofsafety concerns due to its enzyme-inducing and reproductive-toxicactions making it unacceptable for use in women of reproductive age(Dashwood, 1998, Chem Biol Interact. 110:1-5; and Gao et al., 2002,Toxicol Appl Pharmacol. 183:179-88).

One prominent product derived from I3C, and also present in cruciferousplants is 3,3′-diindolylmethane (DIM), the linear dimer molecule formedfrom the condensation of two molecules of I3C. Once formed, DIM isstable in acid and less enzyme inducing than other I3C products(Bradfield et al., 1987, J Toxicol Environ Health. 21:311-23). In cellculture, DIM has been shown to have apoptosis promoting effects in bothestrogen-dependent and independent breast cancer cells (Hong et al.,2002, Biochem Pharmacol 63:1085-97). DIM has also been shown tospecifically induce apoptosis in papillomavirus altered cervical cancercell lines (Chen et al., 2001, J Nutr. 131:3294-302). In animals, orallyadministered DIM inhibits the growth of certain chemically induced formsof breast cancer (Chen et al., 1998, Carcinogenesis, 19:1631-9).

Investigations of DIM have resulted in U.S. Pat. No. 5,948,808,“Indole-3-carbinol, diindolylmethane and substituted analogs asantiestrogens”, which provides for a method of treatingestrogen-dependent cancer. U.S. Pat. No. 6,656,963, “Indole-3-carbinol(I3C) derivatives and methods”, discloses additional derivatives of I3Cfor use in methods to inhibit cancerous cell growth, but specificallyexcludes DIM.

Previous experimental work by the present inventor has described the useof DIM and the related trimeric derivative of I3C,2-(Indol-3-ylmethyl)-3,3′-diindolylmethane (LTR), in specializedformulations (U.S. Pat. No. 6,086,915) and in treatments for breast pain(mastalgia) and endometriosis (U.S. Pat. No. 6,689,387). Endometriosisis a disorder of uterine epithelial tissue (endometrium) which migratesoutside the endometrial cavity of the uterus and resumes growth,typically in the abdomen. Pending applications of the present inventoralso include uses of DIM and LTR for cervical dysplasia (U.S. patentapplication Ser. No. 10/616,477), and for Human Papilloma Virus (HPV)infections (U.S. patent application Ser. No. 10/616,477). Likeendometriosis, cervical dysplasia and HPV infections are disorders ofepithelial tissue and not smooth muscle. Leiomyomas are a distinctpathobiologic entity, arising only in uterine or extra-uterine smoothmuscle which is a distinct tissue with different appearance, structure,function and embryologic origin than endometrium and other epithelialtissue.

A more recent patent application (U.S. Patent Application PublicationNo. 2003/0220377, filed May 7, 2003) describes synthetic indoleanti-estrogenic compounds, structurally unrelated to DIM, which areproposed to treat both cancerous and benign conditions involving theuterus. In distinction to these synthetic, indole anti-estrogens, DIMhas been shown to have estrogenic, growth promoting activity in breastcancer cells (Riby et al., 2000, Biochem Pharmacol. 60:167-77) andestrogen-receptor activating activity in endometrial cancer cells (Leonget al., 2004, Mol. Endocrinol. 18:291-302. Epub 2003 Nov. 26). Finally,U.S. Patent Application Publication No. 2001/0002393 (filed Dec. 20,2000), describes methods and kits for treating and diagnosing leiomyomasusing inhibitors of metalloproteinase enzymes.

It would be beneficial to have new therapeutic options for leiomyomatreatment that are non-invasive, that avoid or minimize surgery, andthat avoid the side effects of systemically administered hormonaltherapies. New medical therapies for the treatment leiomyoma-relatedconditions are needed.

3. SUMMARY OF THE INVENTION

The present invention provides compositions and methods of usingdiindolylmethane and diindolylmethane-related indoles to treat intra-and extra-uterine leiomyomas. The methods typically involve oral use of3,3′-diindolylmethane (DIM) in an effective amount to reverse or retardthe growth of leiomyomas. The methods, using pharmaceutically acceptableformulations, result in a reduction of the size of the myomas and aresolution of myoma-related symptoms. In certain embodiments, thesemethods employ structurally-related, synthetically-derived, substituteddiindolylmethane compounds. Preferred DIM-related compounds for use inthe methods and compositions of the invention include, but are notlimited to, hydroxylated DIMs, methoxylated DIMs,2-(Indol-3-ylmethyl)-3,3′-diindolylmethane (LTR), hydroxylated LTRs,methoxylated LTRs, 5,5′-dimethylDIM (5-Me-DIM), 2,2′-dimethylDIM(2-Me-DIM), 5,5′-dichloroDIM (5-Cl-DIM),imidazolelyl-3,3′-diindolylmethane, nitro-substitutedimidazolelyl-3,3′-diindolylmethanes,2,10-dicarbethoxy-6-methoxy-5,7-dihydro-indolo-[2,3-b]carbazole,6-ethoxycarbonyloxy-5,7-dihydro-indolo-[2,3-b]carbazole and2,10-dicarbethoxy-6-ethoxycarbonyloxy-5,7-dihydro-indolo-[2,3-b]carbazole,and 2,6-dicarbethoxy-3,3′-dimethyl-13,14-diindolylmethane.

In preferred embodiments, DIM or the DIM-related indole is suspended asmicroparticles in a starch carrier matrix.

In certain embodiments, the methods and compositions of the inventioninvolve the oral use of DIM and DIM-related compounds. In certainembodiments, DIM or the DIM-related indole is administeredintra-arterially, vaginally, or is injected directly into myoma tissue.

In other embodiments, the compositions of the present invention usefulin treating leiomyoma-related disease also include suspensions of DIMand related synthetic diindolylmethanes suitable for direct injectioninto leiomyoma tissue at the time of pelvic abdominal surgery or duringtrans-vaginal intra-uterine surgery (hysteroscopy). DIM releasingmicrospheres are also described for use during Uterine ArteryEmbolization (UAE) procedures.

In further embodiments, topical preparations of DIM and relateddiindolylmethanes are described for use in vaginal suppositories totreat leiomyomas. Vaginal suppository applications of DIM and relatedsynthetic diindolylmethanes can be used alone or in conjunction withoral dosage forms for leiomyoma treatment.

In certain embodiments of the invention, the subject is a human. Humanleiomyomas can be uterine leiomyomas or extra-uterine leiomyomas. Inother embodiments, the subject is an avian. Suitable avians include, butare not limited to, chickens and quails. Avian leiomyomas are generallyoviduct leiomyomas.

In another embodiment, the present invention describes oral use of DIMand related synthetic diindolylmethanes in conjunction with orallyactive antagonists of cellular growth factor receptors. This includescombined use of DIM with epidermal growth factor receptor (EGFR)antagonists. EGFR antagonists appropriate for combined use with DIMand/or synthetic derivatives of DIM, includes tyrosine-kinase EGFRinhibitors, such as ZD1839 (Gefitinib, Iressa®, [AstraZeneca, UK]),OSI-774 (Erlotinib, Tarceva®, [OSI Pharmaceuticals, Boulder, Colo.]), CI1033 [Parke-Davis Pharmaceutical Research, Ann Arbor, Mich.], PKI 166[Novartis Pharma, AG (Basel, Switzerland)] and others (e.g., GW2016;N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methylsulfonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine).Other suitable EGFR receptor antagonists include those of natural originwith EGFR inhibiting activity such as silibinin,(−)-epigallocatechin-3-gallate (EGCG), resveratrol, and their cellgrowth inhibiting metabolites or derivatives.

In yet another embodiment, combination therapy for leiomyomas, includedwithin the scope of this invention, includes the use of DIM andDIM-related compounds used with and without EGFR antagonists inconjunction with Uterine Artery Embolization (UAE), surgical myomectomy,and/or low-dose external beam radiation therapy. The object of combineduse of DIM and DIM-related compounds with other treatment modalities forleiomyomas is to increase the efficacy over a single modality therapyalone.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B Reduction of leiomyoma tumor volume in patients treated withoral DIM. A. Subject A. B. Subject B.

5. DETAILED DESCRIPTION OF THE INVENTION

The present inventor has discovered that administration of DIM to afemale or male having leiomyoma tissue results in retarding the growthand reducing the size of myoma tissue. In humans, myoma-associatedsymptoms, including pain, painful or excessive menstrual bleeding, andmyoma-related bowel and urinary dysfunction improve following DIM-basedtreatment. Although rare, leiomyomas also develop outside the uterus inboth women and men. Extra-uterine sites of leiomyoma occurrence includethe nipple, esophagus, scrotum, and seminal vesicles which all possesssmooth muscle (Farman, 1975, S Afr Med J. 49:1333-40). In avian species,DIM, or a DIM-related indole, is used to prevent or delay thedevelopment of leiomyomas of the oviduct and magnum, lengthening theperiod of breeding and egg production in economically important poultry.

5.1. Diindolylmethane and Diindolylmethane Related Indoles

In certain embodiments, the DIM compounds useful in the methods of theinvention include DIM (3,3′-diindolylmethane) and the related linear DIMtrimer (2-(indol-3-ylmethyl)-3,3′-diindolylmethane [also written: 2(Indol-3-ylmethyl)-indol-3-yl]indol-3-ylmethane] (LTR). As used herein,“DIM-related compound”, “DIM-related indole”, and “DIM derivative” areused interchangeably, and refer to both natural metabolites and analogsof DIM, and also to “structurally-related, synthetically-derived,substituted diindolylmethane compounds” and “synthetic derivatives ofDIM”, such as those disclosed herein and known in the art. One ofordinary skill in the art will recognize that in any of thepharmaceutical compositions or methods of the invention where DIM isused, a DIM-related compound, including a structurally-related,synthetically-derived, substituted diindolylmethane compound orsynthetic derivative of DIM, can be used.

The chemical structure of a DIM is as follows (where each of the Rgroups is H):

The chemical structure of LTR is as follows (where each of the R groupsis H):

In certain embodiments, an active hydroxylated or methyoxylatedmetabolite of DIM, i.e., a compound of formula I, wherein R₃₂, R₃₃, R₃₆,and R₃₇ are substituents independently selected from the groupconsisting of hydrogen, hydroxyl, and methoxy, and R₃₁, R₃₄, R₃₅, R₃₈,R₄₁, R₄₂, R₅₀, and R₅₁ are hydrogen, is utilized.

In certain embodiments, an active hydroxylated or methyoxylatedmetabolite of LTR, i.e., a compound of formula II, wherein R₆₂, R₆₃,R₆₆, R₆₇, R₇₀, and R₇₁ are substituents independently selected from thegroup consisting of hydrogen, hydroxyl, and methoxy, and R₆₁, R₆₄, R₆₅,R₆₈, R₆₉, R₇₂, R₈₁, R₈₂, and R₈₃ are hydrogen, is utilized.

In an alternative embodiment, active DIM derivatives with R₃₂ and R₃₆substituents made up of ethoxycarbonyl groups, and R₅₀, R₅₁ are eitherhydrogen or methyl, are utilized. In another embodiment, activesubstituted DIM derivatives including methylated and chlorinatedcompounds, exemplified by those that include 5,5′-dimethylDIM(5-Me-DIM), 2,2′-dimethylDIM (2-Me-DIM), and 5,5′-dichloroDlM (5-C1-DIM)are described in U.S. Patent Application Publication No. 2002/0115708 bySafe, published Aug. 22, 2002, incorporated herein by reference in itsentirety, are utilized in the present invention. In another embodiment,active DIM derivatives include imidazolelyl-3,3′-diindolylmethane,including nitro substituted imidazolelyl-3,3′-diindolylmethanes, andadditional DIM-related compounds described in U.S. Patent ApplicationPublication No. 2004/0043965 by Jong, Ling, published Mar. 4, 2004,incorporated herein by reference in its entirety, are utilized.

In certain embodiments, a DIM related compounds has formula (III):

wherein:

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are substituentsindependently selected from the group consisting of hydrogen, C₁-C₂₄alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl, C₅-C₂₀ aryl, C₆-C₂₄ alkaryl,C₆-C₂₄ aralkyl, halo, hydroxyl, sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀ aryloxy, acyl, acyloxy, C₂-C₂₄alkoxycarbonyl, C₆-C₂₀ aryloxycarbonyl, halocarbonyl, C₂-C₂₄alkylcarbonato, C₆-C₂₀ arylcarbonato, carboxy, carboxylato, carbamoyl,mono-(C₁-C₂₄ alkyl)-substituted carbamoyl, di-(C₁-C₂₄ alkyl)-substitutedcarbamoyl, mono-substituted arylcarbamoyl, thiocarbamoyl, carbamido,cyano, isocyano, cyanato, isocyanato, isothiocyanato, azido, formyl,thioformyl, amino, mono- and di-(C₁-C₂₄ alkyl)-substituted amino, mono-and di-(C₅-C₂₀ aryl)-substituted amino, C₂-C₂₄ alkylamido, C₆-C₂₀arylamido, imino, alkylimino, arylimino, nitro, nitroso, sulfo,sulfonato, C₁-C₂₄ alkylsulfanyl, arylsulfanyl, C₁-C₂₄ alkylsulfinyl,C₅-C₂₀ arylsulfinyl, C₁-C₂₄ alkylsulfonyl, C₅-C₂₀ arylsulfonyl,phosphono, phosphonato, phosphinato, phospho, phosphino, andcombinations thereof, and further wherein any two adjacent (ortho)substituents may be linked to form a cyclic structure selected fromfive-membered rings, six-membered rings, and fused five-membered and/orsix-membered rings, wherein the cyclic structure is aromatic, alicyclic,heteroaromatic, or heteroalicyclic, and has zero to 4 non-hydrogensubstituents and zero to 3 heteroatoms; and

R¹¹ and R¹² are independently selected from the group consisting ofhydrogen, C₁-C₂₄ alkyl, C₂-C₂₄ alkoxycarbonyl, amino-substituted C₁-C₂₄alkyl, (C₁-C₂₄ alkylamino)-substituted C₁-C₂₄ alkyl, and di-(C₁-C₂₄alkyl)amino-substituted C₁-C₂₄ alkyl,

with the provisos that: at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸,R⁹, R¹⁰, R¹¹ and R¹² is other than hydrogen; and when R¹, R², R³, R⁴,R⁵, R⁶, R⁷, and R⁸ are selected from hydrogen, halo, alkyl and alkoxy,then R¹¹ and R¹² are other than hydrogen and alkyl.

A preferred embodiment includes the use of2,10-dicarbethoxy-6-methoxy-5,7-dihydro-indolo-[2,3-b]carbazole(SRI13668 (SRI Inc., Menlo Park, Calif.)). Additional preferredembodiments include the use of6-ethoxycarbonyloxy-5,7-dihydro-indolo-[2,3-b]carbazole and2,10-dicarbethoxy-6-ethoxycarbonyloxy-5,7-dihydro-indolo-[2,3-b]carbazole(SRI Inc., Menlo Park, Calif.).

In another embodiment, a DIM related compounds has formula (IV):

wherein:

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ are substituents independentlyselected from the group consisting of hydrogen, C₁-C₂₄ alkyl, C₂-C₂₄alkenyl, C₂-C₂₄ alkynyl, C₅-C₂₀ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl,halo, hydroxyl, sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄ alkenyloxy, C₂-C₂₄alkynyloxy, C₅-C₂₀ aryloxy, acyl, acyloxy, C₂-C₂₄ alkoxycarbonyl, C₆-C₂₀aryloxycarbonyl, halocarbonyl, C₂-C₂₄ alkylcarbonato, C₆-C₂₀arylcarbonato, carboxy, carboxylato, carbamoyl, mono-(C₁-C₂₄alkyl)-substituted carbamoyl, di-(C₁-C₂₄ alkyl)-substituted carbamoyl,mono-substituted arylcarbamoyl, thiocarbamoyl, carbamido, cyano,isocyano, cyanato, isocyanato, isothiocyanato, azido, formyl,thioformyl, amino, mono- and di-(C₁-C₂₄ alkyl)-substituted amino, mono-and di-(C₅-C₂₀ aryl)-substituted amino, C₂-C₂₄ alkylamido, C₅-C₂₀arylamido, imino, alkylimino, arylimino, nitro, nitroso, sulfo,sulfonato, C₁-C₂₄ alkylsulfanyl, arylsulfanyl, C₁-C₂₄ alkylsulfinyl,C₅-C₂₀ arylsulfinyl, C₁-C₂₄ alkylsulfonyl, C₅-C₂₀ arylsulfonyl,phosphono, phosphonato, phosphinato, phospho, phosphino, andcombinations thereof, and further wherein any two adjacent (ortho)substituents may be linked to form a cyclic structure selected fromfive-membered rings, six-membered rings, and fused five-membered and/orsix-membered rings, wherein the cyclic structure is aromatic, alicyclic,heteroaromatic, or heteroalicyclic, and has zero to 4 non-hydrogensubstituents and zero to 3 heteroatoms, with the proviso that one butnot both of R² and R⁶ is amino, mono-substituted amino, ordi-substituted amino;

R¹¹ and R¹² are independently selected from the group consisting ofhydrogen, C₁-C₂₄ alkyl, C₂-C₂₄ alkoxycarbonyl, amino-substituted C₁-C₂₄alkyl, (C₁-C₂₄ alkylamino)-substituted C₁-C₂₄ alkyl, and di-(C₁-C₂₄alkyl)amino-substituted C₁-C₂₄ alkyl;

R¹³ and R¹⁴ are defined as for R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸, withthe proviso that at least one of R¹³ and R¹⁴ is other than hydrogen; and

X is O, S, arylene, heteroarylene, CR¹⁵R¹⁶ or NR¹⁷ wherein R¹⁵ and R¹⁶are hydrogen, C₁-C₆ alkyl, or together form ═CR¹⁸R¹⁹ where R¹⁸ and R¹⁹are hydrogen or C₁-C₆ alkyl, and R¹⁷ is as defined for R¹¹ and R¹².

A preferred embodiment includes the use of2,6-dicarbethoxy-3,3′-dimethyl-13,14-diindolylmethane (SRI Inc., MenloPark, Calif.).

In another embodiment, a DIM related compounds has formula (V):

wherein:

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R¹¹, R¹², and X are defined as forcompounds of formula (III); and

R²⁰ and R²¹ are defined as for R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸.

In yet another embodiment, the DIM-related indole is anindole-3-carbinol tetrameric derivative (Brandi et al., 2003, CancerRes. 63:4028-4036).

5.2. Dosage and Administration

DIM or a DIM-related compound may be administered by any means and atany dosage, as described below. The actual administered amount of DIM ora DIM-related compound may be decided by a supervising physician orveterinarian and may depend on multiple factors, such as, the age,condition, file history, etc., of the subject, or patient, in question.

The subject, or patient, to be treated using the methods of theinvention is an animal, e.g., avian (such as a chicken or quail) or amammal, and is preferably human, and can be a fetus, child, or adult. Ina preferred embodiment, the subject is a human female.

A preferred embodiment is the oral administration of DIM or syntheticderivatives in an acceptable formulation for oral administration.Preferably, the diindolylmethane used in the invention has beenprocessed to enhance bioavailability, as is described in U.S. Pat. No.6,086,915; however any suitable preparation of diidolylmethane or of astructurally-related, synthetically-derived, substituteddiindolylmethane, can be used in the methods and compositions of theinvention.

DIM is administered in a dose from 25-600 mg/day or 25-500 mg/dayorally. DIM may be administered once per day, or two or more times perday. Preferably the dose is administered 2-3 times per day. Mostpreferably, the DIM is administered in a formulation designed forenhance oral absorption, e.g., microencapsulated with TPGS (as describedin U.S. Pat. No. 6,086,915). This formulation (25-30% DIM by weight) isadministered in a dose of 100-2000 mg/day, or more preferably at a doseof 300-600 mg, providing 75-150 mgs of actual DIM, given orally twiceper day. As an alternative to DIM, the closely related linear trimer(LTR) may be employed in methods and doses described for DIM as a singleagent for leiomyoma treatment. As a further alternative, the orallyactive DIM derivatives described in U.S. Patent Application PublicationNo. 2004/0043965 may be employed in the present invention as singleagents. Structurally-related, synthetically-derived, substituteddiindolylmethane's, as described by Jong (U.S. Pat. No. 6,800,655 andPatent Application Publication No. 2004/0043965) are administeredaccording to the present invention in an acceptable formulation for oraladministration in a dose of 10-400 mg/day. Preferably, these substituteddiindolylmethanes are administered in an absorption-enhanced formulationat a dose of 50 to 250 mg/day.

In an alternative embodiment, DIM or a structurally-related,synthetically-derived, substituted diindolylmethane can be administeredin the form of a sterile, injectable suspension. The suspension isinjected directly into the leiomyoma tissue at the time of exploratorypelvic abdominal surgery, pelvic laparoscopy or hysteroscopy. Injectionof the leiomyoma tissue is followed post operatively by oral use of DIMin an acceptable formulation and at an effective dose. Such a suspensionconsists of, for example, microcrystalline DIM or structurally-related,synthetically-derived, substituted diindolylmethanes (0.2-2% wt/volume)in a suspension of physiologic salts, and pH adjusters. Particle sizesof DIM or structurally-related, substituted diindolylmethane crystals insuspensions are from 50 to 500 microns in average diameter. pH adjusterssuch as NaOH are added to bring the pH to 7.5-8. Preferably, 1-2 cc ofsuspension containing 10-20 mg of DIM or a substituted diindolylmethane,depending on the size of the myoma, is injected directly into eachleiomyoma.

In alternative embodiments for direct injection into fibroid tissue, DIManalogues, including imidazolelyl-3,3′-diindolylmethane, nitrosubstituted imidazolelyl-3,3′-diindolylmethanes and DIM-relatedcompounds described in U.S. Patent Application Publication No.2004/0043965 by Jong, Ling, published Mar. 4, 2004, can be used inmanufacture of the sterile suspension. Preferably, 1-2 cc of suspensioncontaining 0.001 mg/kg to 100 mg per kg of structurally-related,synthetically-derived, substituted diindolylmethane as described by Jongcan be administered by direct injection into individual fibroid tissuemasses.

In another alternative embodiment, injectable emulsions of DIM or astructurally-related, synthetically-derived, substituteddiindolylmethane can be formulated to overcome the low solubility of DIMin both water and lipid. A specialized micro-emulsion utilizesphospholipids to optimize the solubility of DIM and related compoundsand improve the stability of the emulsion. Preferably, 1-3 cc of asterile, injectable emulsion containing 7-20 mg of DIM or substituteddiindolylmethane is injected into each leiomyoma under direct vision,depending on the size of the myoma.

In another alternative embodiment, DIM or a structurally-related,synthetically-derived, substituted diindolylmethane can be incorporatedwithin bio-compatible, stable microspheres for use during Uterine ArteryEmbolization (UAE). In one preferred use, DIM and/or EGFR-antagonistsare included in the production of hydrophilic, non-resorbable,microspheres produced from an acrylic polymer and impregnated withporcine gelatin. Examples of production techniques for DIM-impregnatedmicrospheres for controlled, targeted embolization of myomas aredescribed in U.S. Pat. No. 5,635,215 and U.S. Patent ApplicationPublication No. 2003/0211165 by Vogel et al., published Nov. 13, 2003,both of which are herein expressly incorporated by reference in theirentireties. Dose ranges for administration of DIM andstructurally-related, synthetically-derived, substituteddiindolylmethanes, when used in microspheres as extended-release drugdelivery devices, are from 25-2000 mgs per embolization treatment. Whenused in conjunction with EGF-antagonist impregnated microspheres, thedose range for administration of DIM or a DIM-related compound is25-1000 mgs and the EGFR-antagonist dose is from 250-1000 mgs perembolization treatment. DIM and/or an EGFR inhibitor containingmicrospheres are ideally used in smaller size (50-500 micron diameters)and dose (less than 500-1000 mg total microsphere weight) than currentinert microspheres which are recommended to be greater than 500 micronin diameter and to be used in doses greater than 1000 mg microsphereweight. Smaller active microspheres containing DIM and/or an EGFRinhibitor result in sub-total arterial blockage and reducepost-treatment side effects associated with ischemic tissue necrosis.

Following myomectomy, UAE, or low-dose radiation therapy, typical dosesof oral DIM or a structurally related, synthetically-derived,substituted diindolylmethane are from 25-100 mg twice a day for 2-4months. In an alternative embodiment, oral DIM and a structurallyrelated, synthetically-derived, substituted diindolylmethane can beadministered with orally active EGFR-antagonists. In one embodiment, DIMor a DIM-related compound is used with IRESSA® (Gefitinib [ZD1839]) for1-4 months using an IRESSA dose of 50-100 mg/day.

In an alternative embodiment, DIM or a DIM-related compound can besuspended in a vaginal formulation such as a suppository for use, e.g.,in myoma patients. Typically the suppository is placed high in thevagina once daily providing a dose from 250-1000 mg of DIM. In apreferred embodiment, once daily use of a 500 mg DIM vaginal suppositoryis continued for 2-6 months and accompanied by oral use of an absorptionenhanced formulation of DIM in a dose of 300 mg twice daily providing 75mg of DIM orally, twice daily.

5.3. Combination Therapy

As indicated, the methods and compositions of the present invention arealso useful in combination with other therapeutic agents and therapeuticmodalities which may be used for the treatment of leiomyomas. Acombination of agents is expected to result in more effective therapy tobe used for a shorter duration of treatment. Cell culture study of myomacells using a polymerase chain reaction (PCR) demonstrated an excess inlevels of epidermal growth factor (EGF) messenger RNA which was seen inproliferative phase tissue samples, compared to proliferative phasesamples from normal uteri (Harrison-Woolrych et al., 1994, J ClinEndocrinol Metab. 78:1179-84). These results establish the dominantcontribution of progesterone and elevations of EGF in the abnormalgrowth of myoma tissue.

5.3.1. EGFR Inhibitors

The EGFR inhibitors for use in the methods and compositions of thepresent invention include, but are not limited to, small molecule drugswhich inhibit one or more EGFRs, monoclonal antibodies inactivatingEGFRs, and antisense DNA or RNA inactivating EGFR DNA or RNA deliveredto a cell using gene therapy. EGFRs which may be inhibited include anyEGFR known in the art. See, e.g., Rajkumar, 2001, Current Science81:535-541. Due to expected synergistic interaction of DIM, or astructurally-related DIM, with an EGFR inhibitor, reduced doses of anEGFR inhibitor can be used when used in combination with DIM.

In one embodiment, absorption-enhanced DIM or a structurally-related,synthetically-derived, substituted diindolylmethane is administered inconjunction with an orally active antagonist of the EGF receptor (EGFR).In a preferred embodiment, effective doses of DIM would be the same asused when DIM is administered alone. A representative dose is 15 mg DIM(60 mg of a typical formulated DIM) or 25 mg DIM (100 mg of a typicalformulated DIM).

Small molecular EGFR inhibitors suitable for use in the inventioninclude the EGFR inhibitors, Gefitinib(N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-amine,Iressa, AstraZeneca, UK) and related compounds (see European PatentApplication No. 0566226; International Patent Applications WO 96/33980and WO 97/30034; Woodburn et al., 1997, Proc. Amer. Assoc. CancerResearch 38:633; and Woodburn et al., 1999, Pharmacol. Ther. 82,241-250), Erlotinib(N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-yl]-(3-ethynylphenyl)amine,Tarceva®, OSI Pharmaceuticals [Boulder, Colo.]) and related compounds(see International Patent Applications WO 96/30347 and WO 99/55683), CI1033(6-acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)quinazolin-4-amine,Pfizer [New York, N.Y.]) and related compounds (see International PatentApplications WO 97/38983 and WO 00/31048, and Smaill et al., 1999, J.Med. Chem. 42:1803-1815), PKI 166(4-[(1R)-1-phenylethylamino]-6-(4-hydroxyphenyl)-7H-pyrrolo[2,3-d-]pyrimidine,Novartis Pharma, AG [Basel]) and related compounds (see InternationalPatent Application WO 97/02266) and GW2016(N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methylsulfonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine).

The specific EGFR's to be inhibited include the HER family of EGFR's,PDGFR (Platelet Derived Growth Factor Receptor), and VEGFR (VascularEndothelial-derived Growth Factor Receptor). Representative specificsmall molecule drugs useful in the present invention, presented inrelation to the EGFR inhibited are summarized in Table 1.

TABLE 1 Orally active, GRF Inhibitor Drugs for Use with DIM-RelatedIndoles: HER EGFR Drug Manufacturer Drug Class I II IV IV VEGFR PDGFZD1839 AstraZeneca Small Head Group X Gefitinib Quinazoline (Iressa)(reversible) ZD6474 AstraZeneca X X OSI-774 OSI/Roche/ Small Head GroupX Erlotinib Genentech Quinazoline (Tarceva) (reversible) LapatinibGlaxoSmithKline Large Head Group X X GW-572016 Quinazoline GW-2016GlaxoSmithKline X X STI-571 Novartis X X Imatinib Myesylate (Gleevec)EKB-569 Wyeth (irreversible) X X CI-1033 Pfizer 4-anilinoquinazoline X XX X (PD183805) (irreversible) Cancertinib SU5416 Sugen indolin-2-ketoneX Semaxanib Pharma/Pfizer SU11248 Sugen indolin-2-one X X Pharma/PfizerSU6669 Sugen Pharma X Vatalanib Novartis/Schering anilino- X X X X X XPTK787 phthalazines (ZK222584) PKI-166 Novartis Pyrrolopyrimidines X X X(reversible) CEP-7055 Sanofi-Synthelab Dimethylglycene X X

Representative specific EGFR inhibiting monoclonal antibodies useful inthe present invention presented in relation to the EGFR inhibited,include those that appear in Table 2.

TABLE 2 Representative EGFR-specific antibodies Drug Manufacturer ClassHER VEGFR PDGF Cetuximab ImClone/B-MS Mouse/ X (Erbitux) human mAbTrastuzumab Genentech/DNA mAb X (Herceptin) MDX-210 Medarex mAb XABX-EGF Abgenix/ mAb X Immunex TheraCIM YM mAb EGFR Panitumumab AbBenixmAb EGFR EMD-72000 Merck mAb EGFR bevacizumab DNA/Hoffman mAb X(Avastin) Ranibizumab DNA/Novartis mAb X (Lucentis)

Doses of an EGFR inhibitor on the order of ⅕ the dose when administeredalone can be employed. Typically, Iressa® (Gefitinib) would be employedin a dose of 50-500 mg/day, more preferably, 50-250 mg/day, or 50-100mg/day, as the EGF receptor antagonist. Alternatively, a low, effectivedose of another growth factor antagonist such as OSI-774 (Erlotinib,Tarceva®), CI 1033 [Parke-Davis Pharmaceutical Research (Ann Arbor,Mich.), PKI 166 [Novartis Pharma, AG (Basel, Switzerland)] or GW2016would be employed at doses of 25-500 mg/day. Using combined DIM/EGFRantagonist treatment permits a shortened treatment interval of 1-3months. As an example of such combined therapy, an absorption-enhancedformulation of DIM in a dose of 300 mg [75 mg actual DIM] is takenorally twice daily along with a dose of 100 mg of Iressa (ZD1839,Gefitinib) taken once daily. Alternatively, oral absorption-enhanced DIMformulations or DIM-related derivatives can be used with a member of thetyrosine-kinase inhibitor class of EGF inhibitors, such as ZD1839(Gefitinib, Iressa), OSI-774 (Erlotinib, Tarceva), CI-1033, and GW2016,using effective oral doses of the DIM-related compound and theEGF-antagonist.

Further details of the clinical use of EGF antagonists for combined usewith DIM and/or DIM-related compounds are described in the followingpublications, incorporated herein by reference in its entirety (Janmaatet al., 2003, Oncologist 8:576-86; and Janmaat et al., 2003, Drugs Today(Barc) 39 Suppl C:61-80).

TABLE 3 Dose Ranges for Combined uses of DIM-Related Indoles and EGFRInhibitors Minimal Average Maximal Effective Tolerated Tolerated DoseDose Dose Range Range Range (MED) (ATD) (MTD) Drug Manufacturer mg/daymg/day mg/day Formulated BioResponse  25-150 150-500 500-1000 DIM(BR-DIM) ZD1839 AstraZeneca  25-150 150-350 350-750 Gefitinib (Iressa)Lapatinib GlaxoSmith- 175-500 500-900 900-1,800 GW- Kline 572016 OSI-774OSI/DNA/  50-150 150-200 200-400 Erlotinib Roche (Tarceva) ImatinibNovartis 100-300 300-400 400-800 Myesylate (Gleevec) STI-571 CI-1033Pfizer  10-100 100-500 500-700 Efalizumab  5-25  25-75  75-200 XanelinEKB-569 PKI-166 Novartis  10-50  50-100 100-900 Semaxanib Sugen  10-50 50-100 100-200 SU5416 Pharma/ mg/m² mg/m² mg/m² Pfizer CEP-7055 Sanofi- 25-100 100-400 400-1000 Synthelab

Alternatively, the combinations of DIM (or a DIM-related compound) andan EGFR antagonist of natural origin allows use of EGFR antagonists atmaximal oral dose, due to greater safety and tolerability of EGFRinhibitors of plant origin. Examples of natural polyphenolic compoundswith demonstrated inhibition of EGFR inhibitory activity includeSilibinin, a flavolignan isolated from the fruits of Silibum marianum(Milk Thistle)(Silybin[3,5,7,-trihydroxy-2-[3-(4-hydroxy-3-methoxyphenil)-2-hydroxymethyl-1,4-benxodioxan-6-il]-chronan-4-one];Sharma et al., 2001, Mol. Carcinog. 30:224-36),(−)-epigallocatechin-3-gallate (EGCG), a polyphenolic catechin found ingreen tea (Sah et al., 2004, J Biol. Chem. 279:12755-62) andresveratrol, a stillbene derivative commonly isolated from grapevines(Vitis vinifera L) and from Polygonium cuspidatum Sieb. Et Zucc(Japanese knotweed). Also useful are compounds related to resveratrolsuch as viniferins, piceatannol (3,4,3′,5′-tetrahydroxystilbene),oxyresveratrol (2,3′,4,5′-tetrahydroxystilbene), 4,4′-dihydroxystilbene,and cis- and trans-piceids which are also preferred for use in thepresent invention (Stewart et al., 2004, Invest. New Drugs 22:107-117).Doses of compounds related to resveratrol to be used in the methods andcompositions of the invention are similar to doses used for resveratrol.In certain embodiments, silibinin, EGCG, or resveratrol can beincorporated with DIM-related indoles in vaginal suppositories fortopical application in proximity to uterine leiomyomas.

TABLE 4 Dose Ranges for Combined uses of DIM-Related Indoles and EGFRInhibitors of Natural Origin Effective Dose Drug Manufacturer Range(mg/day) Formulated BioResponse,  25-750 DIM LLC (BR-DIM) SilibininIndena, Inc. 100-2000 (SiliPhos) Resveratrol Interpharma 100-2000(RegrapeX) Praha EGCG Various 500-2000

Also useful in combination with DIM, or DIM-related indole, in treatingLeiomyomatous conditions are extracts of Scutellaria barbata D. Don(SB), an herbal component of the traditional Korean medicine known as‘Ban-Ji-Ryun,’ which demonstrate growth inhibitory activity in leiomyomacell culture (Lee et al., 2004, Int Immunopharmacol. 4:447-54) andEvodiamine, an indole alkaloid component extracted from the fruit ofEvodiae Fuctus (Evodia rutaecarpa Benth). The Scutellaria extract istypically given at a dose of 200-800 mg/day, and the Evodia extract istypically given in a dose of 300-800 mg/day, providing Evodiamine(40-200 mg/day).

Since the 4-hydroxy estrogen metabolites characteristics of leiomyomatissue are pro-oxidants, selected anti-oxidant compounds are of furtherbenefit for use in combined therapy with DIM. These include Lycopene,the major carotenoid found in tomatoes. Lycopene has demonstratedinhibition of leiomyoma-related smooth muscle tumors in the oviducts ofJapanese quail (Sahin et al., 2004, Nutr Cancer.50:181-9). In addition,lycopene, and related beta-carotene, protect cells from the pro-oxidanteffects of 4-hydroxy estrogen metabolites, known to be produced inleiomyoma tissue (Muzandu et al., 2005, Jpn J Vet Res. 52:173-84).Typically, Lycopene is administered in association with a DIM relatedindole in a daily dose range from 4-30 mg/day. Lycopene or a relatedcarotenoid can also be incorporated in vaginal suppositories for topicalapplication to the uterus via the uterine cervix.

The combinations of DIM (or a DIM-related compound) and an EGFRantagonist, with or without a carotenoid or Scutellaria extract, can bein the same composition for administering in combination concurrently,or in different compositions for administering concurrently butseparately, or sequentially. When the compounds are administeredsequentially, the compounds can be administered within several minutes,several hours or several days of each other.

The combined use of DIM, or a structurally-related,synthetically-derived, substituted diindolylmethane, and anEGFR-antagonist can be further combined with other interventionalmodalities of leiomyoma treatment. Interventional modalities oftreatment preferably include myomectomy, UAE, and low dose radiationtherapy. Myomectomy is performed during pelvic laparotomy, pelviclaparoscopy, or uterine hysteroscopy. Myoma tissue not amenable toremoval during these procedures and so is injected directly during theprocedure with suspensions of DIM, or a DIM related compound, or withsuspensions of microspheres impregnated with DIM or a DIM relatedcompound, optionally with an EGFR-antagonist. Surgery is followed byoral administration of DIM, or a DIM-related compound, alone or inconjunction with EGFR-antagonist. UAE can be performed using thestandard administration of non-resorbable acrylic microspheres(Embosphere Microspheres [Biosphere Medical, Inc., Rockland, Mass.])followed by 1-4 months of oral administration of DIM or a DIM-relatedcompound alone or in conjunction with EGFR-antagonist such as IRESSA. Inan alternative embodiment, UAE is performed utilizing a microsphere asdescribed in Example 5 manufactured to contain and slowly release DIM ora structurally-related, synthetically-derived, substituteddiindolylmethane. To administer DIM containing microspheres, a sterilesuspension of microspheres is first mixed with non-ionic contrast media.Typically, a total dose of 1-3 cc of a suspension of microspheres havinga diameter of 300-500 microns which contains 250-750 mgs of DIM, or astructurally-related, synthetically-derived, substituteddiindolylmethane, is mixed with contrast media and injected into one orboth uterine arteries using fluoroscopic guidance. Preferably, DIMimpregnated microsomes would be used in combination with EGFR-antagonistimpregnated microsomes to take advantage of anticipated synergisticgrowth inhibitory activities of the two therapeutic agents. Finally, theavailability of lesion directed radiation therapy permits combined useof DIM and a structurally-related, synthetically-derived, substituteddiindolylmethane during low dose radiation therapy to take advantage ofanticipated radiation sensitizing activity of the DIM or DIM-relatedcompounds and EGFR antagonists. In this combined use, an oral DIMformulation with or without an EGFR-antagonist is administered before,during, and after a series of radiation therapy treatments. In apreferred embodiment, “Gammaknife” or “Cyberknife” (Accuray, Inc.,Sunnyvale, Calif.) radiation therapy technology is used to concentrateand focus the radiation beam limiting the radiation exposure ofadjacent, bladder, ovarian, and intestinal tissue. Non-invasiveradiation therapy treatment, limited to the myoma, is followed by oraltreatment with oral DIM, structurally-related, synthetically-derived,substituted diindolylmethane's, optionally with IRESSA or other orallyactive EGF antagonist. Preferably, the cGy dose is held below thethreshold dose known to cause damage to adjacent structures. A computerprogram such as the one described in U.S. Pat. No. 6,477,229 can be usedto choose a safe cumulative radiation dose. Following Cyberkniferadiation treatment, an absorption-enhanced formulation of DIM in a doseof 300 mg [75 mg actual DIM] is taken orally twice daily alone or with adose of 50-100 mg of Iressa (ZD1839, Gefitinib) taken once daily.

5.4. Pharmaceutical Compositions

The pharmaceutical compositions according to the present inventionpreferably comprise one or more pharmaceutically acceptable carriers andthe active constituents. The carrier(s) must be “acceptable” in thesense of being compatible with the other ingredients of the compositionand not deleterious to the recipient thereof.

It will be appreciated that the amounts of DIM or a DIM-related compoundrequired for said treatment will vary according to the route ofadministration, the disorder to be treated, the condition, age, and filehistory of the subject, the galenic formulation of the pharmaceuticalcomposition, etc.

Preferably, the DIM or a DIM-related compound used in the invention hasbeen processed to enhance bioavailability, as is described in U.S. Pat.No. 6,086,915. DIM or LTR processed in this manner is referred to as“processed DIM” and “processed LTR”, respectively. However, any suitablepreparation of DIM or a DIM-related compound can be used in the methodsand compositions of the invention.

The following is a list of ingredients useful for formulating processedDIM or a DIM-related compound:

1. About 10 to about 40 percent by weight of DIM or a DIM-relatedcompound.

2. About 10 to about 40 percent by weight of the following, alone or incombination: vitamin E succinate polyethylene glycol 1000; vitamin Esuccinate Polyethylene glycols with polyethylene glycol (with amolecular weight range of 400-2000); other polyethylene glycol esterssuch as those formed by fatty acids such as oleic acid or stearic acid;polyvinylpyrrolidones; polyvinylpolypyrrolidones; Poloxamer 188, Tweens;or Spans.

3. About 5 to about 20 percent by weight of the following, alone or incombination: phosphatidyl choline (derived from soy lecithin andsupplied as Phospholipon 50G from Rhone Poulenc Rorer); dioleoylphosphatidylcholine; phoshatidylglycerol; dioleoylphosphatidylglycerol;dimyristoylphosphatidylcholine; dipalmitoylphosphatidylcholine;phosphatidylethalolamines; phosphatidylserines; or sphingomyelins; orother sources of phospholipids as those from purified Milk Fat GlobuleMembrane; glycerolesters; poly glycerol esters; or ethoxylated castoroil.

4. About 15 to about 30 percent by weight of the following, alone or incombination: hexanol; ethanol; butanol; heptanol; 2-methyl-1-pentanol;various ketone solvents that would be acceptable in foods such as methylethyl ketone, acetone and others; propylene glycol; and certain estersolvents such as ethyl acetate.

5. About 20 to about 40 percent by weight of the following, alone or incombination: modified starch such as Capsul™ Starch from NationalStarch, Inc.; methylcellulose; hydroxypropyl methylcellulose;hydroxyethylcellulose; hydroxypropylethylcellulose; pectin; gum arabic;gelatin; or other polymeric matrix-forming preparation known to thoseskilled in the art, soluble in water and, suitable for spray drying.

6. About 0.5 to about 35 percent by weight of the following, alone or incombination: aerosil 200; or any other flow enhancing excipient fromsilica, or related salt, known to those skilled in the art.

The following is a detailed method of formulating processed DIM:

1. 6.75 kg of TPGS is heated just beyond its melting point with constantstirring in a heated vessel (“First vessel”).

2. 9.38 kg of hexanol and 9.83 kg of jet milled DIM is added to thefirst vessel and the mixture stirred to a uniform suspension at 70° C.1.4 kg of phosphatidyl choline is then added.

3. In a second larger vessel, 185 L of water and 10.7 kg of starch arethoroughly mixed with a Cowles blade. This mixture is neutralized to pH7 with a small amount of sodium carbonate and then heated to 75° C. andstirred for 1 hour.

4. The contents of the first vessel is added to the starch mixture inthe second larger vessel and thoroughly mixed with a homogenizingrotor/stator type mixer at moderate speed for 15 minutes.

5. The mixture from step 4 is spray dried with a small amount(approximately 0.5%) of hydrophilic silica to provide a free flowingpowder of finely dispersed microparticles containing the co-precipitatedTPGS, phosphatidyl choline and DIM in an amorphous, non-crystallinestructure.

6. The flowable powder product is collected and stored in evacuated foilsacks, after de-aerating and flushing with nitrogen.

7. Analysis of presence of unchanged dietary ingredient, reveals about30 to about 33 percent by weight of DIM.

The procedure of making processed DIM may be summarized as follows:

-   -   (a) heating one or more solubilizing emulsifiers selected from        the group consisting of vitamin E succinate polyethylene glycol        1000, polyvinylpyrrolidone, polyoxyethylene stearate, sodium        cholate, deoxycholate and taurocholate;    -   (b) adding to the product of step (a) a solvent and a surfactant        phospholipid selected from the group consisting of phosphatidyl        choline, dioleoyl phosphatidyl choline, phosphatidylglycerol,        dioleoylphosphatidylglycerol, dimyristoylphosphatidylcholine,        dipalitoylphosphatidylcholine, phosphatidylethanolamine,        phosphatidylserine and sphingomyelin to produce a solution;    -   (c) dissolving in the solution of step (b) LTR and/or DIM;    -   (d) adding to the solution of step (c) a solution containing an        encapsulator;    -   (e) mixing the solution produced in step (d) to produce a        microdispersion with a particle size of 5 microns or less; and    -   (f) spray drying the resulting mixture to leave a solid        hydrophobic phytochemical composition.

In general, a suitable (therapeutically effective) amount of DIM or LTRis 50-500 mg per day. DIM is preferably administered in an absorptionenhancing formulation, as described in U.S. Pat. No. 6,086,915, at50-200 mg per day, more preferably 400-800 mg per day, as a suspensionof microparticles in a starch carrier matrix. The LTR is preferablyadministered in an absorption enhancing formulation at 50-200 mg perday, more preferably 200-800 mg per day, as a suspension ofmicroparticles in a starch carrier matrix. The actually administeredamounts of phytochemical may be decided by a supervising physician orveterinarian.

Therapeutic formulations include those suitable for parenteral(including intramuscular and intravenous), intra-arterial, oral, rectal,intra-vaginal, or intradermal administration, although oraladministration is the preferred route. Thus, the pharmaceuticalcomposition may be formulated as parenteral suspension, degradable andnon-degradable microspheres for intra-arterial administration duringUAE, tablets, pills, syrups, capsules, suppositories, formulations fortransdermal application, formulations for intradermal uses, powders,especially lyophilized powders for reconstitution with a carrier forintravenous administration, feed pellets for veterinary use, etc.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which the therapeutic is administered. The carriers in thepharmaceutical composition may comprise a binder, such asmicrocrystalline cellulose, polyvinylpyrrolidone (polyvidone orpovidone), gum tragacanth, gelatin, starch, lactose or lactosemonohydrate; a disintegrating agent, such as alginic acid, maize starchand the like; a lubricant or surfactant, such as magnesium stearate, orsodium lauryl sulphate; a glidant, such as colloidal silicon dioxide; asweetening agent, such as sucrose or saccharin; and/or a flavoringagent, such as peppermint, methyl salicylate, or orange flavoring.

Therapeutic formulations suitable for oral administration, e.g., tabletsand pills, may be obtained by compression or molding, optionally withone or more accessory ingredients. Compressed tablets may be prepared bymixing DIM or a structurally-related, synthetically derived, substituteddiindolylmethane, and compressing this mixture in a suitable apparatusinto tablets having a suitable size. Prior to the mixing, the DIM orstructurally-related DIM may be mixed with a binder, a lubricant, aninert diluent and/or a disintegrating agent.

In a preferred embodiment, DIM or a structurally-related, syntheticallyderived, substituted diindolylmethane is mixed with a binder, such asmicrocrystalline cellulose, and a surfactant, such as sodium laurylsulphate until a homogeneous mixture is obtained. Subsequently, anotherbinder, such as polyvidone, is transferred to the mixture under stirringwith a small amount of added water. This mixture is passed throughgranulating sieves and dried by desiccation before compression intotablets in a standard tableting apparatus.

A tablet may be coated or uncoated. An uncoated tablet may be scored. Acoated tablet may be coated with sugar, shellac, film or other entericcoating agents.

Therapeutic formulations suitable for parenteral administration includesterile solutions or suspensions of the active constituents. An aqueousor oily carrier may be used. Such pharmaceutical carriers can be sterileliquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil and the like. Formulations for parenteral administrationalso include a lyophilized powder comprising phytochemical that is to bereconstituted by dissolving in a pharmaceutically acceptable carrierthat dissolves said phytochemical.

When the pharmaceutical composition is a capsule, it may contain aliquid carrier, such as a fatty oil, e.g., cacao butter.

Suitable pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. These compositions cantake the form of solutions, suspensions, emulsion, tablets, pills,capsules, powders, sustained-release formulations and the like. Thecomposition can be formulated as a suppository, with traditional bindersand carriers such as triglycerides.

In yet another embodiment, the therapeutic compound can be delivered ina controlled release system. In one embodiment, a pump may be used (seeLanger, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201;Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J.Med. 321:574). In another embodiment, polymeric materials can be used(see Medical Applications of Controlled Release, Langer and Wise (eds.),CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, NewYork (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem.23:61 (1983); see also Levy et al., 1985, Science 228:190; During etal., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg.71:105).

For use in UAE and myoma tissue injection, stable, biocompatiblemicrospheres, easily injected through needles and angiography catheters,are preferred. Microspheres with diameters from 10 to about 300micrometers in diameter are most preferred. Due to the poor watersolubility of DIM and structurally-related, synthetically-derived,substituted diindolylmethanes, a method for preparation of biodegradablepolymeric drug delivery devices using relatively low temperatures andnon-aqueous solutions is useful. Techniques for manufacture ofmicrospheres appropriate for the physicochemical characteristics of DIM,LTR, and synthetic DIM-related drugs are described in U.S. Pat. No.5,718,921, which is incorporated by reference herein in its entirety.Manufacture of appropriate microspheres with desirable sustained releasecharacteristics containing DIM or a structurally-related,synthetically-derived, substituted diindolylmethane, and/orEGFR-antagonist is further described in U.S. Patent ApplicationPublication No. 2003/0211165 by Vogel et al., published Nov. 13, 2003,which is incorporated herein by reference in its entirety.Alternatively, embolic compositions comprising macromers having abackbone of a polymer having units with a 1,2-diol and/or 1,3-diolstructure which incorporate DIM or a structurally-related,synthetically-derived, substituted diindolylmethane, and/orEGFR-antagonists can be made according to U.S. Patent ApplicationPublication No. 2003/0223956 by Goupil et al., published Dec. 4, 2003,which is incorporated herein by reference in its entirety.

In other embodiments, a controlled release formulation comprisingbiodegradable polymer microspheres or microparticles wherein DIM or astructurally-related, synthetically-derived, substituteddiindolylmethane is suspended in a polymer matrix, the polymer matrixbeing formed from at least two highly water soluble biodegradablepolymers, and the microspheres being coated with a (d, 1lactide-glycolide) copolymer is preferred. The selection of theparticular (d, 1 lactide-glycolide) copolymer will depend in a largepart on how long a period the microsphere is intended to release theactive ingredient. For example, a (d, 1 lactide-glycolide) copolymermade from about 80% lactic acid and 20% glycolic acid is very stable andwould provide a microsphere suitable for release of DIM, LTR, andsynthetic DIM-related drugs over a period of weeks.

Other controlled release systems are discussed in the review by Langer(1990, Science 249:1527-1533).

In one embodiment of the pharmaceutical composition according to theinvention, two or more active constituents are comprised as separateentities.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients forpracticing the methods of the invention. Optionally associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration.

The invention is further explained by the following illustrativeexamples.

6. EXAMPLE 1 Manufacture of an Absorption-Enhanced Formulation of Dimfor Treatment of Leiomyomas

In general, a suitable (therapeutically effective) amount ofdiindolylmethane is preferably administered in an absorption enhancingformulation, as described in U.S. Pat. No. 6,086,915, at 100-2000 mg perday as a suspension of microparticles in a starch carrier matrix. Theactually administered amounts of Diindolylmethane may be decided by asupervising physician.

Preparation of Processed Diindolylmethane was Accomplished According tothe steps outlined in U.S. Pat. No. 6,086,915, herein incorporated byreference in its entirety. Briefly, this included mixture of about10-40% by final weight of either diindolylmethane with about 10-40% byfinal weight of vitamin E polyethylene glycol 1000 succinate(Vitamin-E-TPGS, Eastman Chemical), 2-20% by final weight, phosphatidylcholine (Phospholipon 50G, Rhone Poulenc) and 15-30% by final weighthexanol. This mixture was made homogeneous by mixing. The homogeneousmixture of indoles and other oil soluble substituents listed above wasadded to a solution of modified starch in water (Capsul Starch fromNational Starch, Inc.). The starch component forms from 30-70% of thefinal dry weight of the product. The well dispersed final combinedmixture was then subjected to spray drying. The resultant product was afine powder containing either diindolylmethane contained within thestarch particles.

Alternatively, the starch component described above as Capsul Starchfrom National Starch, Inc., can instead be made using Maltodextrin NF(Maltrin M100, GPC), 20-30% of the final dry weight, together with GumArabic USP/NF (TIC Pretested, TIC Gums), 20-30% of the final dry weight,together making 40-70% of the final dry weight. Additionally, fumedsilica (Aerosil 200, Degussa), 1-2% of the final weight, can be addedduring the spray drying process as a flow aid.

7. EXAMPLE 2 Manufacture of Capsules Containing Diindolylmethane

Capsules containing 150-300 mg of processed diindolylmethane, asproduced according to the steps described in Example 1, were made bymixing the processed diindolylmethane with microcrystalline celluloseand placing the mixed powder into opaque gelatin capsules.

Alternatively, capsules containing processed diindolylmethane (DIM), asproduced according to the steps described in Example 1, were made bymixing the processed DIM with polyphenolic EGFR inhibitory compoundsaccording to the following per capsule amounts. The ingredients aremixed according to these amounts and capsules are filled using standardmachinery.

TABLE 5 Preferred Range of Capsule Capsule Component Weight (mg) Weight(mg) Processed DIM 75  (50-100) Silibinin (SiliPhos) 100 (100-200) GreenTea Extract (EGCG) 150 (150-300) Total Active Fill Weight (mg) 325(300-625)

Optionally, 6-25 mg of Lycopene is added to each capsule and/or 25-100mg of Scutellaria barbata or Evodia rutaecarpa extract is additionallyadded as desired by the formulator. 1-6 capsules of the combinedformulation are taken, preferably twice daily, with water as advised bya health care practitioner or label instructions.

8. EXAMPLE 3 Manufacture of a Vaginal Suppository Formulation of Dim forTreatment of Leiomyomas in Conjunction with Oral Therapy

In a heated vessel, 90 grams cetostearyl alcohol (Alfol 16/18, Vista)was heated to 100° C. to which 5 gms of microcrystalline DIM (LKT Labs,St. Paul, Minn.) was added with constant mixing to form a hot slurry.Alternatively, 90 grams cetostearyl alcohol (Alfol 16/18, Vista) isheated to 100° C. to which 5 gms of microcrystalline DIM is mixed, aloneor together with 10 grams of ceramide or synthetic cerimide derivatives,C2 ceramide. In a second vessel 400 gms of IV Novata (Semi-syntheticGlyceride Suppository Base, Ashland Chemicals) was warmed to 40° C. withconstant mixing. The well mixed slurry from the first vessel was addedwith continued mixing to the second vessel. The homogenized moltedsuppository material was formed into suppositories of 2 gms each andcooled. Glyceryl monsterate 10-50 gms was added to the molten mixture asneeded to increase the firmness of the final suppositories.

Alternatively, vaginal suppositories are produced using microcrystallineDIM as described above, combined with silibinin, resveratrol, orpharmaceutical EGFR antagonist (e.g., Gefitinib) to provide a minimallyeffective dose for the particular EGFR inhibitor (25-500 mg) per 2 gmsuppository.

9. EXAMPLE 4 Manufacture of an Injectable Emulsion Formulation of Dimfor Treatment of Leiomyomas

To prepare a DIM emulsion for injection into leiomyomas, Ethyl oleate(EO), Phosphatidyl Choline (PC) (from egg yolk), and calcein, ispurchased from Sigma-Aldrich, Inc (St. Louis, Mo.).Distearoyl-phosphatidylethanolamin-N-poly(ethyleneglycol) 2000(DSPE-PEG) is purchased from Avanti Polar Lipids (Alabaster, Ala.).

Using a modification of the method of Yu et al. (Yu W et al., 1993, Int.J. Pharm. 89:139-146), the microemulsion is manufactured as follows: 160grams of EO and 60 grams of PC are dissolved in 1 liter pure ethanol. 24grams of microcrystalline DIM (mean particle size 0.25 micron) is addedand is dissolved in this “oily phase”. 20 grams of DSPEG-PEG is thendissolved in 500 cc of USP water (Aqueous phase). The oily ethanolicsolution (oily phase) with the dissolved DIM is then slowly added intothe DSPE-PEG solution (aqueous phase) under moderate magnetic stirring.The aqueous phase immediately turns milky with opalescence as the resultof the microemulsion produced. The microemulsion is then subjected tolow pressure at 360 mm Hg and maintained at 50° C. The low pressure isused to concentrate the emulsion through removal of the ethanol and aportion of the water. Using an infrared absorption assay to determinethe DIM content of the microemulsion, a final concentration of DIM of7.5 mg/ml is established. Sodium hydroxide is added to increase the pHto the 5.0-7.5 range.

Using this manufacturing technique emulsions of DIM are prepared and aresubjected to stability testing to demonstrate that the particle sizewithin the emulsion remains between 150 and 200 nm. The productiontechnique results in a micro-emulsion with % weight ranges of thecomponents in the following preferred ranges:

Component Approx % Weight DIM 0.05-0.1 Lipids (EO:PC:DSPE-PEG; 8:3:1)  45-28 Water   50-70 Ethanol   1-2

Alternatively, an ethanol-free production method can be utilized toproduce a stable micro-emulsion of DIM or DIM derivatives and analogues,using Lipofundin MCT B. Braun Melsungen AG (Melsungen, Germany), apreformed basic emulsion, and high pressure homogenization ofmicrocrystalline DIM. This method utilizes jet-milled DIM, with particlesize reduced to 0.1 micron average diameter (performed by MicronTechnologies, Inc., Exton, Pa.). Using this technique 700 mg of 0.1micron diameter DIM crystals are homogenized in 100 cc Lipofundin usingequipment and methods as described (Akkar et al., 2003, Eur J PharmBiopharm. 55:305-12). This will result in a stable lipid-basedmicro-emulsion with particle size less than 200 nm and a DIM content of7 mg/cc of the emulsion.

10. EXAMPLE 5 Manufacture of Stable Microspheres Containing Dim forTreatment of Leiomyomas During Selective Uterine Artery Embolisation(UAE)

Due to the poor water solubility of DIM, a production technique forstable, biocompatible microspheres containing DIM is developed based onproduction technology described in U.S. Pat. No. 5,718,921, hereinexpressly incorporated by reference in its entirety. This process uses apolyanhydride polymer which is dissolved in a volatile organic solvent,in which the DIM or structurally-related, synthetically-derived,substituted diindolylmethane is dispersed and co-dissolved in thepolymer solution. The mixture is suspended in an organic oil, and theorganic solvent is extracted into the oil, creating microspheres. Themethod enables the preparation of DIM containing microspheres from avariety of biodegradable polymers, including hydrophobic polyanhydridessuch as (pCPP:SA, 50:50) and CPP copolymerized with dodecanedoic acid(DD), (pCPP:DD, 20:80) and (pCPP:DD, 50:50).

For the preparation of DIM microspheres, four grams of the polymer,pCPP:SA, 20:80, mw=16000, is dissolved in 20 ml methylene chloride, towhich is added 1 gram of microcrystalline DIM and suspended in thepolymer solution using a mechanical stirrer. The mixture is then droppedinto silicon oil (Dow Chemical Company, Midland, Mich.) that containsbetween approximately 1.0 and 20% of Span™ or another surfactant oremulsifying agent. Span™ emulsifiers are preferred. This is then stirredat a set stirring rate. Stirring is done using an overhead stirrer typeRZR50, (“CAFRAMA”, Wiarton, Ont.) and a three-blade impeller. After 1hour, petroleum ether is introduced and stirring is continued foranother hour. The microspheres are isolated by filtration, washed withpetroleum ether, dried overnight in a lyophilizer (Labconco, FreezeDryer 8), are sieved (U.S. Standard Sieve Series, Newark, Wire ClothCompany, Newark, N.J.) and are stored at less than 0° C.

This process of manufacture will yield DIM impregnated microspheres withdiameters of from 50 to 1000 microns. The recovery in this productionprocess can be limited to 50% due to some polymer precipitating on thestirrer. The DIM microspheres are sieved to remove microspheres withdiameters greater than 500 microns to result in microspheres with afinal size distribution of 50-500 microns. The smaller microspheres arethen sieved to remove those with diameters less than 50 microns, leavingmicrospheres with diameters between 500 and 500 microns. The resulting 2grams of microspheres are again dried in a lyophilizer, autoclaved tosterilize, and resuspended at a concentration of 1.0 ml of microspheresin 5 ml of sterile physiological saline. The 5 ml suspension ofmicrospheres will contain approximately 250 mgs of DIM, adequate fortherapeutic use in a single UAE procedure.

11. EXAMPLE 6 Use of Oral, Absorption-Enhanced Dim for the Treatment ofLeiomyomas in a Woman

Application of orally active formulations of DIM or synthetic DIMderivatives is illustrated by the following clinical example ofsuccessful treatment of a myomatos condition.

A 44-year old female was symptomatic from uterine leiomyoma,experiencing painful menses with excessive bleeding, frequency ofurination, and inability to exercise due to tiredness. Physicalexamination by her gynecologist revealed a right pelvic mass visible oninspection of the abdomen and palpable with gentle physical exam. Herpelvic exam revealed visible and palpable distortion of the uterinecervix due to protruding leiomyoma tissue. Bimanual palpation revealed aclearly enlarged uterus with an irregular contour consistent withmultiple leiomyomas. A blood test indicated mild anemia. Hysterectomywas strongly suggested. The patient initiated therapy with capsules ofDIM, formulated for enhanced absorption as described in Example 1. Adose of 120 mg of DIM formulation, given three times per day, delivering30 mg of actual DIM per dose, was used. After two months of DIM use, thepatient returned to her physician describing normal menstrual flow withnormal levels of discomfort and improved energy. Her repeat physicalexam included a normal abdominal exam with no visible or palpable pelvicmasses. Her repeat pelvic exam showed a normal appearing uterine cervixand a palpable uterus of much reduced size.

12. EXAMPLE 7 Use of Oral, Absorption-Enhanced Dim to Reduce the Size ofUterine Leiomyomas

Clinical treatment and laboratory study are being used to demonstratethe therapeutic benefits of oral DIM for leiomyoma patients associatedwith a reduced production of 4-hydroxy estrogen metabolites. Expectedresults are clinical improvement in symptoms and reduction of leiomyomasize documented in serial sonograms. Clinical treatment, using oral DIMformulated for enhanced absorption according to Example 1, hasdemonstrated meaningful reduction of leiomyoma size demonstrated byserial pelvic ultrasound evaluation.

Women with uterine leiomyomas have been selected based on the findingsof abnormal uterine sonograms showing measurable leiomyoma documented onmore than one occasion. Three subjects were proceeding to provide abaseline 12-hour urine collection and began to take absorbable DIM 150to 450 mg [37.5-112 mg actual DIM] twice daily for 3-6 months. Two womenhave completed participation in a preliminary study, completing 2 and 6month treatment periods. Subject A was a 46 yr old multiparous femalewith regular menstrual periods, taking thyroid hormone replacement tocorrect primary hypothyroidism and using no other medications. Subject Bwas a 50 year old female taking no medications with regular periods.

After informed consent, a pretreatment ultrasound was obtained for eachsubject. Each subject provided a 12 or 24 hour urine specimen which wasfrozen. Subject A began taking 2 capsules twice daily ofabsorption-enhanced DIM (BioResponse, LLC, Boulder Colo.) providing DIMat 100 mg/kg per dose. Subject B took 3 capsules twice daily ofabsorption-enhanced DIM (BioResponse, LLC, Boulder Colo.) providing DIMat 150 mg/kg per dose. After 2 months of treatment, Subject A wasre-evaluated by the same ultrasound technician using the same equipmentand at the same phase of her menstrual cycle. A post treatment urine wasalso obtained, collected at the same point in the menstrual cycle as thebaseline urine sample. Similarly, Subject B was re-evaluated after 6months of treatment using absorption-enhanced DIM (BioResponse-DIM). Shetook a higher twice daily dose providing 150 mg/kg/dose. Herre-evaluation included pelvic ultrasonography following a series of 3pre-treatment pelvic ultrasound evaluations all performed with the sameequipment. Like Subject A, Subject B provided a before and after DIMtreatment urine sample.

The pelvic ultrasound data were analysed using a mathematical formula tocalculate the pre-treatment and post-treatment volume of individualleiomyoma based on the widest diameter and the diameter perpendicular tothis measured in centimeters (cm). A calculation of individual tumorvolume was performed, treating each tumor as an ovoid mass, using theformula: V=4/3π(H/2)²(L/2), where V=volume in cubic centimeters (cm³),H=tumor height in centimeters (cm), and L=tumor length in centimeters(cm). This method provided individual tumor volumes in cubic centimeters(cm³). Results for the pre and post treatment tumor volumes with percentreduction following the 2 month treatment of Subject A and the 6 monthtreatment of Subject B are presented in Table 6 and 7 and FIGS. 1A-B.The average percent reduction in tumor volume was 33.5% for Subject A.The average reduction in tumor volume was 78.6% for Subject B.

TABLE 6 Leiomyoma Tumor Volumes in Subject “A” Before and After DIMTreatment Tumor Pre-DIM Post-DIM Percent Identification TreatmentTreatment Decrease in Number Tumor Volume Tumor Volume Tumor Subject “A”(cm³) (cm³) Volume Tumor 1 22.39327 3.783001 83.10653 Tumor 2 116.8987114.4901 2.060378 Tumor 3 62.83185 53.20497 15.32167

TABLE 7 Leiomyoma Tumor Volumes in Subject “B” Before and After DIMTreatment Tumor Pre-DIM Post-DIM Percent Identification TreatmentTreatment Decrease in Number Tumor Volume Tumor Volume Tumor Subject “B”(cm³) (cm³) Volume Tumor 1 19.8836635 5.57528 71.9605 Tumor 2 3.5913641.876578 47.74749 Tumor 3 9.91067762 6.093643 38.51437 Tumor 41.88495559 0.884882 53.05556 Tumor 5 2.11848065 0.796394 62.40732

The urine samples were tested at the Bradlow/Sepkovic Laboratory,Hackensack University Medical Center, Hackensack, N.J. The techniqueutilized was an established gas chromatography-mass spectrometry (GCMS)assay for estradiol (E2), estrone (E1), 2-hydroxy estrone (2OH-E1),4-hydroxy estrone (4OH-E1), 16-hydroxy estrone (16OH-E1), and estriol(E3) in urine (Michnovicz et al., 1997, J Natl Cancer Inst.89(10):718-23). The procedure involved overnight incubation withglucuronidase to glucouroides and subsequent analysis by derivitasationand GCMS determination of nanogram per milliliter of urine per 24 hours(ng/ml/24 hr). Laboratory analysis is expected to show an increase in2-hydroxy estrone, and a decrease in 16-hydroxy estrone, as measured inpre-treatment as compared to post-treatment urine samples. A decrease in4-hydroxy estrone would be consistent with inhibition of estrogenmetabolism characteristic of leiomyomas (Liehr et al., 1995, Proc NatlAcad Sci USA. 92:9220-4). Results of before and after DIM treatment arepresented in Table 8, showing the metabolites of interest as a percentof the total estrogen metabolites measured and as ratios of the amountsof 2OH-E1 to 16OH-E1 (2OH-E1/16OH-E1) determined for each sample.Comparison of results revealed large treatment related increases in2OH-E1 and the 2OH-E1/16OH-E1 ratio in both Subjects. Subject A showed atreatment related reduction in 16OH-E1 and Subject B, treated at ahigher dose, showed a treatment related reduction in 4OH-E1. 2OH-E1estrogen metabolites are non-growth promoting, and weaker estrogenscompared to 16OH-E1 and 4OH-E1.

TABLE 8 Percent or Ratio of Measured Estrogen Metabolites Subject (DoseEstrogen Metabolite or Before After and Duration) Metabolite Ratio DIMDIM “A” 2OH-Estrone 4% 8.3% (200 mg/day DIM, (2OH-E₁) Taken for 24OH-Estrone 0.4% 0.7% Months) (4OH-E₁) 16OH-Estrone 8.4% 4.9% (16OH-E₁)2OH-E₁/16OH-E₁ 0.5 1.7 “B” 2OH-Estrone 2.5% 35% (300 mg/day DIM,(2OH-E₁) Taken for 6 Months) 4OH-Estrone 2.5 1.7% (4OH-E₁)2OH-E₁/16OH-E₁ 0.3 9.5

In conclusion, preliminary treatment of women with oral DIM resulted ina consistent reduction of intra-uterine leiomyoma size. Unlike the useof injected GnRN agonists (Flierman et al., 2005, BJOG. 112:638-42), orlong acting progestins (Venkatachalam et al., 2004, J Obstet. Gynaecol.24:798-800), the reduction in leiomyoma volume seen with DIM treatmentwas not associated with amenorrhea, hypo-estrogenemia, hot flashes, orother reported side effects.

Further prospective, placebo-controlled clinical study of oral DIM inselected Leiomyoma patients will be used to further establish theutility of DIM-related methods of leiomyoma treatment.

13. EXAMPLE 8 Organ Culture of Uterine Leiomyoma Tissue Treated with Dimand/or Egf Antagonists

An in vitro study of the effects of DIM and EGFR inhibitors will beundertaken. A protocol to establish the activity and synergism of DIM,and/or Gefitinib (Iressa®, ZD1839 [Astra Zeneca]) and other EGFRinhibitors, based on the exposure of primary cultures of leiomyomatissue, is designed. These studies will utilize sex steroid supportedgrowth of leiomyoma tissue in organ culture as a pre-clinical model.Iressa® is an orally active EGFR-TKI (epidermal growth factor receptortyrosine kinase inhibitor) which blocks signal transduction pathwayswhich may contribute to leiomyoma growth. Other inhibitors of theepidermal growth factor receptor (EGFR) to be tested include CI-1033[Parke-Davis Pharmaceutical Research (Ann Arbor, Mich.)], a quinazolinetyrosine kinase inhibitor different from Iressa, and PKI 166 [NovartisPharma, AG (Base1)], a non-quinazoline EGFR antagonist. The effects ofDIM alone and in combination with an EGFR antagonist on leiomyoma cellgrowth are evaluated using the EVA/PCD (ex vivo apoptotic/programmedcell death) assay (Rational Therapeutics Cancer Evaluation Laboratories,Long Beach, Calif.) which has previously been shown to correlate withresponse, time and survival in patients with certain tumors.

Dose-response curves are interpolated to provide 50% lethalconcentrations (LC(50)). The degree of synergy (by median effect) andnormalised Z-scores (raw scores converted to relative activitydistributed around the mean) is then computed.

Favorable interactions are anticipated for DIM combinations with EGFreceptor antagonists. Leiomyoma cultures will be analyzed forsynergistic increases in apoptosis-related cell killing withcombinations of DIM and EGFR inhibitors. These primary leiomyoma culturestudies may support synergistic and possibly clinically beneficialinteractions of DIM and EGFR inhibitors.

Leiomyoma tissue will be obtained at the time of surgery from womenconsenting to be donors of their myoma tissue following hysterectomy ormyomectomy utilizing the Institutional Review Board. Fresh samples ofleiomyoma tissue will be initiated in organ culture followingestablished protocols in the laboratories of Rational Therapeutics, LongBeach, Calif. Published techniques of tissue culture for normal uterinemyometrium and leiomyoma tissue include those described by Horiuchi etal. (Horiuchi et al., 2000, Mol Hum Reprod. 6:523-8), and Arici et al.(Arici et al., 2003, Am J Obstet Gynecol. 188(1):76-83). Cultureconditions will include the following:

-   -   myoma tissue plus estrogen    -   myoma tissue plus estrogen and progesterone    -   myoma tissue, estrogen, progesterone, and DIM    -   myoma tissue, estrogen, progesterone, and Iressa    -   myoma tissue, estrogen, progesterone, DIM and Gefitinib (Iressa,        AstraZeneca, UK)    -   myoma tissue, estrogen, progesterone, DIM and CI 1033        [Parke-Davis Pharmaceutical Research (Ann Arbor, Mich.)]    -   myoma tissue, estrogen, progesterone, DIM and PKI 166 [Novartis        Pharma, AG (Basel, Switzerland)]    -   myoma tissue, estrogen, progesterone, DIM and Silibinin (LKT        Labs, St. Paul, Minn.)    -   myoma tissue, estrogen, progesterone, DIM and EGCG (LKT Labs,        St. Paul, Minn.)    -   myoma tissue, estrogen, progesterone, DIM and Evodiamine (Sigma,        St. Louis, Mo.)

Cultures will be subjected to the standardized EVA cell death assayperformed by Rational Therapeutics and scored according to rates ofapoptosis. The supernatant samples will be frozen and subsequentlytested at the Bradlow/Sepkovic Laboratory, Hackensack University MedicalCenter, Hackensack, N.J. for estrogen metabolites. The method ofanalysis for supernatants will utilize an established gaschromatography-mass spectrometry (GCMS) assay for the levels of2-hydroxy, 16-hydroxy, and 4-hydroxy estrone in ng/ml of supernatant (Xuet al., 2002, J Chromatogr B Analyt Technol Biomed Life Sci.780:315-30).

14. EXAMPLE 9 In Vivo Experiment in Japanese Quail to DemonstratePrevention of Leiomyomas Using Dim

Introduction:

The animal model of monitored growth of the Japanese quail (Coturnixcoturnix japonica) will be used to demonstrate the utility of DIM, or aDIM-related indole, for the prevention and treatment of spontaneousleiomyomas. Similar to leiomyomas in humans, smooth muscle tumors of theoviduct and the ligament of the oviduct are among the most common benigntumors seen in avian species (Foster et al., 1989, Poult Sci.68:1447-53). The purpose of this experiment is to demonstrateanti-leiomyoma activity of DIM-related indoles in Japanese quail that isamplified by combined treatment with EGFR inhibitors and/or carotenoidantioxidants. The results are directly applicable to humans and avariety of economically important species of birds.

Methods:

Experimental methods for maintenance of quail, feeding, assessment andsampling of oviduct tissue follow published techniques (Sahin et al.,2004, Nutr Cancer. 50:181-9). 200-300 6-month old Japanese quail(Coturnix coturnix japonica) will be used in the study. Groups of 30-40birds will be assigned to the following Experimental Groups:

1. Control Group: Basal Diet (17% crude protein and 12.4 mJ/kgmetabolizable energy)

2. Low Dose DIM Group: Basal Diet plus Formulated DIM (DIM formulated asin Example 1)

3. High Dose DIM Group: Basal Diet plus Formulated DIM

4. Low Dose DIM plus Lycopene: Basal Diet plus Formulated DIM plusLycopene

5. Low Dose DIM plus Silibinin: Basal Diet plus Formulated DIM plusSilibinin

6. Low Dose DIM plus Evodiamine: Basal Diet plus Formulated DIM plusEvodiamine

Birds will receive supplemented diet according to the following doseranges.

TABLE 9 Experimental Number DIM (mg/kg/diet) Lycopene SilibininEvodiamine Group (n) Low Dose High Dose (mg/kg/diet) (mg/kg/diet)(mg/kg/diet) Control (30-40) DIM only (30-40) (25-50) (Low Dose) DIMonly (30-40) (100-200) (High Dose) DIM and (30-40) (25-50) (100-200)Lycopene DIM and (30-40) (25-50) (100-300) Silibinin DIM and (30-40)(25-50) (25-100) Evodiamine

At the end of 270-300 days of treatment, birds will be slaughtered andthe presence or absence and diameter of leiomyomas in the smooth muscleof the oviduct will be recorded. The tumorous and surrounding normalsmooth muscle tissue will be examined histologically. The presence andappearance of leiomyomas will be compared among groups. The tumorous andsurrounding normal smooth muscle will be stained usingimmuno-histochemistry for the presence and levels of phosphorylated Aktand markers of NF kappa b activation using published techniques (Hapmanet al., 2004, J Clin Endocrinol Metab. 89:5683-93).

Expected Results:

Fewer and smaller leiomyomas are expected to be found in DIM and DIMplus EGFR inhibitor treated groups of quail. Reduced levels ofphosphorylated Akt and markers of NF kappa b activation will be found insmooth muscle and leiomyomas of treated versus control birds.

Discussion

Leiomyomas of the uterus represent a major health problem in thedeveloped world and interfere with reproduction and egg laying incommercial avian species. Treatment with DIM-related indoles, with orwithout EGFR and/or carotenoid antioxidants, provides a new approach toprevention and non-surgical treatment of leiomyomas and related, benignsmooth muscle tumors.

The present invention is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the invention, and functionally equivalent methodsand components are within the scope of the invention. Indeed, variousmodifications of the invention, in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and accompanying drawings. Such modifications areintended to fall within the scope of the appended claims.

Various references are cited herein, the disclosure of which areincorporated by reference in their entirety.

1. A method of treating leiomyoma in a human having leiomyoma comprisingadministering to the subject an amount of 3,3′ diindolylmethane (DIM) ora DIM-related indole effective to reduce one or more symptoms associatedwith leiomyoma.
 2. The method of claim 1, wherein DIM is administered.3. The method of claim 1, wherein the DIM-related indole is selectedfrom the group consisting of: a compound of formula I:

wherein R³² and R³⁶ are substituents independently selected from thegroup consisting of hydrogen, hydroxyl, and methoxy, and ethoxycarbonylgroups, R³³ and R³⁷ are substituents independently selected from thegroup consisting of hydrogen, hydroxyl, and methoxy, R³¹, R³⁴, R³⁵, R³⁸,R⁴¹, and R⁴² are hydrogen, and R⁵⁰, R⁵¹ are either hydrogen or methyl; acompound of formula II:

wherein R⁶², R⁶³, R⁶⁶, R⁶⁷, R⁷⁰, and R⁷¹ are substituents independentlyselected from the group consisting of hydrogen, hydroxyl, and methoxy,and R⁶¹, R⁶⁴, R⁶⁵, R⁶⁸, R⁶⁹, R⁷², R⁸¹, R⁸², and R⁸³ are hydrogen; acompound of formula (III):

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are substituentsindependently selected from the group consisting of hydrogen, C₁-C₂₄alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl, C₅-C₂₀ aryl, C₆-C₂₄ alkaryl,C₆-C₂₄ aralkyl, halo, hydroxyl, sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀ aryloxy, acyl, acyloxy, C₂-C₂₄alkoxycarbonyl, C₆-C₂₀ aryloxycarbonyl, halocarbonyl, C₂-C₂₄alkylcarbonato, C₆-C₂₀ arylcarbonato, carboxy, carboxylato, carbamoyl,mono-(C₁-C₂₄ alkyl)-substituted carbamoyl, di-(C₁-C₂₄ alkyl)-substitutedcarbamoyl, mono-substituted arylcarbamoyl, thiocarbamoyl, carbamido,cyano, isocyano, cyanato, isocyanato, isothiocyanato, azido, formyl,thioformyl, amino, mono- and di-(C₁-C₂₄ alkyl)-substituted amino, mono-and di-(C₅-C₂₀ aryl)-substituted amino, C₂-C₂₄ alkylamido, C₆-C₂₀arylamido, imino, alkylimino, arylimino, nitro, nitroso, sulfo,sulfonato, C₁-C₂₄ alkylsulfanyl, arylsulfanyl, C₁-C₂₄ alkylsulfinyl,C₅-C₂₀ arylsulfinyl, C₁-C₂₄ alkylsulfonyl, C₅-C₂₀ arylsulfonyl,phosphono, phosphonato, phosphinato, phospho, phosphino, andcombinations thereof, and further wherein any two adjacent (ortho)substituents may be linked to form a cyclic structure selected fromfive-membered rings, six-membered rings, and fused five-membered and/orsix-membered rings, wherein the cyclic structure is aromatic, alicyclic,heteroaromatic, or heteroalicyclic, and has zero to 4 non-hydrogensubstituents and zero to 3 heteroatoms, and R¹¹ and R¹² areindependently selected from the group consisting of hydrogen, C₁-C₂₄alkyl, C₂-C₂₄ alkoxycarbonyl, amino-substituted C₁-C₂₄ alkyl, (C₁-C₂₄alkylamino)-substituted C₁-C₂₄ alkyl, and di-(C₁-C₂₄alkyl)amino-substituted C₁-C₂₄ alkyl, with the provisos that at leastone of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² is otherthan hydrogen, and when R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ are selectedfrom hydrogen, halo, alkyl and alkoxy, then R¹¹ and R¹² are other thanhydrogen and alkyl; a compound of formula (IV):

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ are substituentsindependently selected from the group consisting of hydrogen, C₁-C₂₄alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl, C₅-C₂₀ aryl, C₆-C₂₄ alkaryl,C₆-C₂₄ aralkyl, halo, hydroxyl, sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀ aryloxy, acyl, acyloxy, C₂-C₂₄alkoxycarbonyl, C₆-C₂₀ aryloxycarbonyl, halocarbonyl, C₂-C₂₄alkylcarbonato, C₆-C₂₀ arylcarbonato, carboxy, carboxylato, carbamoyl,mono-(C₁-C₂₄ alkyl)-substituted carbamoyl, di-(C₁-C₂₄ alkyl)-substitutedcarbamoyl, mono-substituted arylcarbamoyl, thiocarbamoyl, carbamido,cyano, isocyano, cyanato, isocyanato, isothiocyanato, azido, formyl,thioformyl, amino, mono- and di-(C₁-C₂₄ alkyl)-substituted amino, mono-and di-(C₅-C₂₀ aryl)-substituted amino, C₂-C₂₄ alkylamido, C₅-C₂₀arylamido, imino, alkylimino, arylimino, nitro, nitroso, sulfo,sulfonato, C₁-C₂₄ alkylsulfanyl, arylsulfanyl, C₁-C₂₄ alkylsulfanyl,C₅-C₂₀ arylsulfinyl, C₁-C₂₄ alkylsulfonyl, C₅-C₂₀ arylsulfonyl,phosphono, phosphonato, phosphinato, phospho, phosphino, andcombinations thereof, and further wherein any two adjacent (ortho)substituents may be linked to form a cyclic structure selected fromfive-membered rings, six-membered rings, and fused five-membered and/orsix-membered rings, wherein the cyclic structure is aromatic, alicyclic,heteroaromatic, or heteroalicyclic, and has zero to 4 non-hydrogensubstituents and zero to 3 heteroatoms, with the proviso that one butnot both of R² and R⁶ is amino, mono-substituted amino, ordi-substituted amino; R¹¹ and R¹² are independently selected from thegroup consisting of hydrogen, C₁-C₂₄ alkyl, C₂-C₂₄ alkoxycarbonyl,amino-substituted C₁-C₂₄ alkyl, (C₁-C₂₄ alkylamino)-substituted C₁-C₂₄alkyl, and di-(C₁-C₂₄ alkyl)amino-substituted C₁-C₂₄ alkyl, R¹³ and R¹⁴are defined as for R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸, with the provisothat at least one of R¹³ and R¹⁴ is other than hydrogen, and X is O, S,arylene, heteroarylene, CR¹⁵R¹⁶ or NR¹⁷ wherein R¹⁵ and R¹⁶ arehydrogen, C₁-C₆ alkyl, or together form ═CR¹⁸R¹⁹ where R¹⁸ and R¹⁹ arehydrogen or C₁-C₆ alkyl, and R¹⁷ is as defined for R¹¹ and R¹²; and acompound of formula (V):

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R¹¹, R¹², and X are defined asfor compounds of formula (III), and R²⁰ and R²¹ are defined as for R¹,R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸.
 4. The method of claim 1, wherein theDIM-related indole is selected from the group consisting of hydroxylatedDIMs, methoxylated DIMs, 2-(Indol-3-ylmethyl)-5 3,3′-diindolylmethane(LTR), hydroxylated LTRs, methoxylated LTRs, 5,5′-dimethylDIM(5-Me-DIM), 2,2′-dimethylDIM (2-Me-DIM), 5,5′-dichloroDIM (5-CI-DIM),imidazolelyl-3,3′-diindolylmethane, nitro-substitutedimidazolelyl-3,3′-diindolylmethanes,2,10-dicarbethoxy-6-methoxy-5,7-dihydro-indolo-[2,3-b]carbazole,6-ethoxycarbonyloxy-5,7-dihydro-indolo-[2,3-b]carbazole and2,10-dicarbethoxy-6-ethoxycarbonyloxy-5,7-dihydro-10indolo-[2,3-b]carbazole, and2,6-dicarbethoxy-3,3′-dimethyl-13,14-diindolylmethane.
 5. The method ofclaim 1, wherein the DIM or DIM-related indole is suspended asmicroparticles in a starch carrier matrix.
 6. The method of claim 1,wherein the DIM or DIM-related indole is administered orally.
 7. Themethod of claim 1, wherein the DIM or DIM-related indole is administeredintra-arterially.
 8. The method of claim 1, wherein the DIM orDIM-related indole is administered vaginally.
 9. The method of claim 1,wherein the DIM or DIM-related indole is injected directly into myomatissue.
 10. The method of claim 1, wherein the leiomyoma is an uterineleiomyoma.
 11. The method of claim 1, wherein the leiomyoma is anextra-uterine leiomyoma.
 12. The method of claim 1, further comprisingadministering an EGFR antagonist.
 13. The method of claim 12, whereinthe EGFR antagonist is gefitinib.
 14. The method of claim 12, whereinthe EGFR antagonist is silibinin, (−)-epigallocatechin-3-gallate, orresveratrol.